Fig. 5

YAP is a critical mediator of TGF-β1/SMAD2-induced EMT and cell invasion. a The effect of TGF-β1 (10 ng/ml) on BxPC-3-shYAP and BxPC-3-shNC PC invasion capability was assessed using a Matrigel invasion assay. PCs with or without TGF-β1 (10 ng/ml) pretreatment for 48 h were seeded into Matrigel-coated invasion chambers. b The invasive cells were quantified by counting the number of cells in 10 random fields at 100 × magnification. c BxPC-3-shYAP and BxPC-3-shNC PCs were pretreated with or without TGF-β1 (10 ng/ml) for 6 h, and cells were stained with an anti-Smad2 antibody (red) and counterstained with DAPI (blue) to identify nuclei (bar, 50 μm). Representative images demonstrate that knockdown of YAP not only reduced TGF-β1-induced nuclear accumulation of Smad2 but also diminished whole–cell Smad2 levels. d BxPC-3-shYAP and BxPC-3-shNC PCs were pretreated with or without TGF-β1 (10 ng/ml) for 48 h, and then, western blot assays were performed to evaluate the expression of E-cadherin, N-cadherin, Vimentin and YAP at the protein level. e BxPC-3 cells were treated with TGF-β1 (10 ng/ml) for the indicated time (10 min, 30 min, 1 h, 3 h and 6 h), and western blot assays were performed to evaluate the expression of YAP and t-SMAD2 and the p-SMAD2 level. f BxPC-3-shYAP and BxPC-3-shNC PCs were pretreated with or without TGF-β1 (10 ng/ml) for 1 h, and then, western blot assays were performed to evaluate the p-SMAD2 and YAP level. g BxPC-3 cells were treated with TGF-β1 at the indicated concentration (1, 2, 5, 10 and 20 ng/ml) for 24 h, and western blot assays were performed to evaluate the protein expression of YAP. h BxPC-3 cells were pretreated with the specific TGF-β1 inhibitor SB431542 (10 μM) for 1 h and then treated with TGF-β1 (10 ng/ml) for 24 h, and western blot assays were performed to evaluate the protein expression of YAP. Column: mean; bar: SD; *P < 0.05; **P < 0.01